PROJECT SUMMARY Gliomas are aggressive brain cancers. Treatment is surgical resection of the tumor supplemented by radio and chemotherapy. Patient prognosis is best when gross total tumor resection is achieved. However, gross total resection is a challenge for gliomas that diffuse extensively and microscopically into the adjacent normal brain parenchyma. Neurosurgeons augment their ability to visualize tumorous tissue though imaging technology like MRI, 5ALA (contrast agent), and/or OCT (optical coherence tomography technique to visualize cancer) that enhances contrast of physical structures associated with the diseased tissue. Because details and spatial information are limited, especially when approaching the periphery of the tumor, there is significant need for the development of alternative approaches that can complement imaging modalities by providing microscopic observation of pathological features of tissue and guide resection maneuvers; there is a need to determine intraoperative pathological molecular characteristics to determine how thorough the resection should be and/or future local therapies to be considered at the time of surgery. The traditional approach to assess tissue pathology is the optical observation of cellular morphology through microscopy but it has limited applicability during surgery because it requires time-consuming laboratory testing and it gives the surgeon no intraoperative molecular information. We propose the use of desorption electrospray ionization mass spectrometry (DESI-MS) as an intraoperative pathology tool to provide diagnostic and prognostic information that can improve the surgical decision-making process. DESI-MS detects the underlying molecular changes occurring in cancerous cells and tissue and can do so within minutes working on minimally processed tissue smears. DESI sprays tissue with a mist of charged droplets to generate and then mass-measure ions. We target 2-hydroxyglutarate to assess the presence of IDH mutations in the tissue and membrane phospholipids and N-acetylaspartate to estimate tumor infiltration (i.e. tumor cell percentage), at locations of particular interest to the surgeon. Neither measurement is provided in current surgical or diagnostic protocols, but these compounds are markers associated with improved surgical outcomes and best patient prognosis. This proposal seeks to test intraoperative DESI-MS during surgical glioma resection cases executed at Mayo Clinic under the supervision of PIs Quiones-Hinojosa and Chaichana to (i) further the development of the DESI-MS technology, the feasibility of which was demonstrated in a preliminary intraoperative study conducted at Indiana University School of Medicine under PI Cooks, (ii) collect an extensive set of data that will allow us to strongly link intraoperative molecular findings with preoperative imaging and postoperative diagnostics for validation, and (iii) set the foundation of follow-up clinical studies measuring prognostic utility.